Identification of broad-spectrum antiviral compounds and assessment of the druggability of their target for efficacy against respiratory syncytial virus (RSV)

Abstract

The search for novel therapeutic interventions for viral disease is a challenging pursuit, hallmarked by the paucity of antiviral agents currently prescribed. Targeting of viral proteins has the inextricable challenge of rise of resistance. Safe and effective vaccines are not possible for many viral pathogens. New approaches are required to address the unmet medical need in this area. We undertook a cell-based high-throughput screen to identify leads for development of drugs to treat respiratory syncytial virus (RSV), a serious pediatric pathogen. We identified compounds that are potent (nanomolar) inhibitors of RSV in vitro in HEp-2 cells and in primary human bronchial epithelial cells and were shown to act postentry. Interestingly, two scaffolds exhibited broad-spectrum activity among multiple RNA viruses. Using the chemical matter as a probe, we identified the targets and identified a common cellular pathway: the de novo pyrimidine biosynthesis pathway. Both targets were validated in vitro and showed no significant cell cytotoxicity except for activity against proliferative B- and T-type lymphoid cells. Corollary to this finding was to understand the consequences of inhibition of the target to the host. An in vivo assessment for antiviral efficacy failed to demonstrate reduced viral load, but revealed microscopic changes and a trend toward reduced pyrimidine pools and findings in histopathology. We present here a discovery program that includes screen, target identification, validation, and druggability that can be broadly applied to identify and interrogate other host factors for antiviral effect starting from chemical matter of unknown target/mechanism of action.

Broad-spectrum inhibition of common respiratory RNA viruses by a pyrimidine synthesis inhibitor with involvement of the host antiviral response

“…Our study into the mechanism of action of this compound indicates that FA-613 interferes with the cellular de novo pyrimidine biosynthesis pathway. Broad-spectrum antiviral activity of pyrimidine synthesis inhibitors has been identified previously and their antiviral effect has been mainly attributed to the depletion of the nucleosides necessary for replication of the viral genome [18][19][20]. Surprisingly, however, we found a connection between the antiviral activity of FA-613 and the use of cells that lack a functional interferon pathway.…”

“…FA-613 interferes with pyrimidine metabolism Several broad-spectrum antivirals were reported to be pyrimidine synthesis inhibitors through inhibition of dihydroorotate dehydrogenase (DHODH) [18][19][20], the fourth enzyme of the de novo pyrimidine biosynthesis pathway (Fig. 3a), and it has been speculated that the inhibition activity of these compounds is a direct consequence of the deprivation of the nucleosides necessary for virus replication.…”

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

“…Optional: supplement culture medium with uridine at 20 μg/ml. Note: When screening chemical libraries for viral replication inhibitors, it seems that compounds targeting early steps of pyrimidine biosynthesis pathway are frequently isolated 13,16,27,28 . The addition of uridine to culture medium is used to filter out such antiviral molecules.…”

Activation de la réponse innée antivirale par des inhibiteurs de la biosynthèse des pyrimidines

“…Alors que nous testions sans succès différentes hypothèses, plusieurs groupes ont publié simultanément des travaux montrant que des molécules inhibitrices de la voie de biosynthèse des pyrimidines sont des antiviraux à large spectre [19][20][21][22]. Même si rien ne suggérait un lien fonctionnel entre cette voie métabolique et la réponse innée antivirale, nous avons testé la capacité du composé DD264 à bloquer la biosynthèse de novo des pyrimidines (Figure 3).…”